Preparing Drosophila Samples for SDS-PAGE - (Apr/15/2005 )

I'm looking for some opinions on what is the best way to prepare drosophila for an SDS-PAGE gel.

The last time i did it, i ground about 50 flies up in 2x extraction buffer then i boiled for 5 mins and spun for about the same (i think...)

Anyone have any suggestions? Improvements to this?

There's got to be other Drosophilists out there:)

~QG

-QueensGal-

oops, i mean i used 2x sample buffer, not extraction buffer!

~QG

-QueensGal-

could you send a brief description of your protocol (you don't have to mention the very specific details if you don't wan't or can't do)?

-Rnilo-

Well, i dont have a formal protocol that i could find so i just asked around and found that was the basic way to extract protein from drosophila for an SDS-PAGE.

I put about 50 flies in a 1.5 eppendorf, added approximately 1mL of 2x sample buffer (pretty standard sample buffer, dont have the recipe offhand). I then boiled for 5 minutes in a water bath and spun down the sample for 5 mins at 14000 rpm.

I then took the supernatant and froze it down.

Thats pretty much it, i just wondered if someone had a better way of doing this!

Thanks for your help:)

~QG

-QueensGal-

hi, i work on drosophila and i'm not sure if this is any use, but...

if you suspend your mashed up flies in a lysis buffer before hand, then spin, you could separate the soluable fraction - where most of the protein is.

-mnqcljsm-

ooh, do you have a protocol for this you could share? I would really appreciate it!

Thanks

~QG

-QueensGal-

QUOTE (mnqcljsm @ Apr 16 2005, 11:07 AM)

hi, i work on drosophila and i'm not sure if this is any use, but...

if you suspend your mashed up flies in a lysis buffer before hand, then spin, you could separate the soluable fraction - where most of the protein is.

I routinely do Westerns from Drosophila heads. I have attached my protocol. I strongly suggest that you do a protein assay for each sample prior to running the Western. If you are trying to compare samples at all, you will need to know how much total protein per sample per lane. Good luck with it! It usu works well for me.

Cheers,

LTR

-L_Reiter-

i have the same protocol and Always work for me...but havent when I tried to do a western using antibodies against histones modifications , didnt work... someone has a protocol to extract histones from drosophila??